Digital PCR Assay for Single-Cell STEC Gene Detection
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Solution Overview
Problem
Existing real-time PCR assays for detecting Enterohemorrhagic Escherichia coli (EHEC) strains in food products suffer from high false-positive rates due to signal interference from other bacteriophages and Gram-negative bacteria, necessitating an assay that can accurately detect target virulence genes in a single cell.
Innovation Solution
A partition-based digital PCR assay using specific primer pairs (e.g., SEQ ID NO: 1 and SEQ ID NO: 2, SEQ ID NO: 5 and SEQ ID NO: 6, etc.) and modified nucleic acid probes (e.g., SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 7, etc.) with detectable labels to enhance specificity and sensitivity in detecting Shiga toxin-producing E. coli (STEC) strains.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If real-time PCR assay is used to detect EHEC strains, then detection capability is provided, but false-positive results occur due to signal interference from other bacteriophages and Gram-negative bacteria
Solution Approach 1:
The patent applies segmentation by dividing the detection process into multiple independent digital PCR reactions, each targeting specific virulence genes (stx1, stx2, eae). This segmentation allows individual gene detection with high specificity, eliminating cross-reactivity issues that cause false positives in conventional real-time PCR assays.
Solution Approach 2:
The patent uses probe-based detection as an intermediary mechanism between the target DNA and the detection system. The probes are designed to specifically hybridize with target sequences, providing an additional layer of specificity that prevents signal interference from non-target bacteriophages and Gram-negative bacteria.
2Measurement precision
If conventional PCR assays are used, then detection of target genes is achieved, but detection sensitivity at single-cell level is insufficient
Solution Approach 1:
The patent employs digital PCR technology that partitions the sample into numerous individual reactions, enabling detection and quantification at the single-cell level. This segmentation approach provides both high sensitivity for detecting low-abundance targets and reliability through statistical analysis of partition positive rates.
Solution Approach 2:
The patent changes the detection parameter from bulk signal averaging in real-time PCR to single-partition signal detection in digital PCR. This parameter change enables detection sensitivity at the single-cell level while maintaining reliability through the all-or-nothing amplification principle in each partition.
3Productivity
If real-time PCR assays are used for EHEC detection, then screening capability is provided, but false-positive results lead to unnecessary product recalls
Solution Approach 1:
The patent segments the detection into multiple specific gene targets (stx1, stx2, eae) rather than using a single broad-specificity assay. This segmentation maintains high screening efficiency while improving result accuracy by detecting only confirmed virulence genes, reducing false positives that lead to unnecessary recalls.
Solution Approach 2:
The patent replaces the mechanical signal detection system of real-time PCR with a digital counting system that enumerates positive partitions. This substitution provides absolute quantification and eliminates the signal interference issues that cause false positives, while maintaining high throughput screening capability.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
The assay effectively discriminates between virulent and avirulent STEC strains, providing accurate detection of target genes like stx and eae directly from a single E. coli cell, reducing false positives and improving detection reliability in food samples.
Implementation Method 1
a target nucleic acid within the bacterial cell is hybridized to at least one primer pair and at least one probe
Implementation Method 2
the probe comprises a modified nucleic acid and at least one detectable label
Data Source
AI summary
The present disclosure relates primer pairs, probes, kits, and methods of use thereof to detect virulent strains of Shiga toxin-producing Escherichia coli (E. coli) (STEC).